Method for Measuring Lipoprotein-Specific Apolipoproteins

Abstract

The present invention is directed to methods of measuring the concentration of lipoprotein particles and/or lipoprotein-specific apolipoproteins in a biological fluid using an immunoassay, without the need of preliminary physical separation of the various types of lipoprotein particles present in the biological fluid.

1 . A method of measuring the concentration of lipoprotein particles in a biological fluid of a subject, wherein an apolipoprotein is structurally associated with said lipoprotein in the form of lipoprotein-apolipoprotein, the method comprising the steps of:
 incubating Solution S2, comprising said biological fluid, a surfactant, and an antibody against said apolipoprotein, for a period of time t2 at a set temperature T2, to generate an antibody-bound apolipoprotein;   generating a calibration curve for quantitating binding of said antibody to said apolipoprotein with an immunoassay, using at least one standard solution comprising a known concentration of said apolipoprotein;   quantitating said antibody-bound apolipoprotein in Solution S2 with said immunoassay using said calibration curve;   determining concentration of said lipoprotein-apolipoprotein in said Solution S2;   determining concentration of said lipoprotein-apolipoprotein in said biological fluid; and,   determining said concentration of said lipoprotein particles in said biological fluid.   
     
     
         2 . The method of  claim 1 , wherein said subject is human. 
     
     
         3 . The method of  claim 1 , wherein said biological fluid is selected from the group consisting of blood, serum and plasma. 
     
     
         4 . The method of  claim 1 , wherein said Solution S2 is generated by a method comprising the steps of:
 mixing said biological fluid with Reagent R1, wherein said Reagent R1 comprises a surfactant, to generate Solution S1;   incubating said Solution S1 for a period of time t1 at a set temperature T1; and,   mixing said Solution S1 with Reagent R2, wherein said Reagent R2 comprises said antibody against said apolipoprotein, to generate said Solution S2.   
     
     
         5 . The method of  claim 1 , wherein said Solution S2 is generated by a method comprising the steps of:
 mixing said biological fluid with Reagent R1, to generate Solution S1;   incubating said Solution S1 for a period of time t1 at a set temperature T1; and,   mixing said Solution S1 with Reagent R2, wherein said Reagent R2 comprises said surfactant and said antibody against said apolipoprotein, to generate said Solution S2.   
     
     
         6 . The method of  claim 1 , wherein said Solution S2 is generated by a method comprising the step of:
 mixing said biological fluid with Reagent R2, wherein said Reagent R2 comprises said surfactant and said antibody against said apolipoprotein, to generate said Solution S2.   
     
     
         7 . The method of  claim 1 , wherein said lipoprotein is LDL and said apolipoprotein is apo B. 
     
     
         8 . The method of  claim 1 , wherein said lipoprotein is HDL and said apolipoprotein is selected from the group consisting of apo A-I, apo A-II, apo A-IV, apo A-V, apo C-I, apo C-II, apo C-III, apo C-IV, and apo E. 
     
     
         9 . The method of  claim 1 , wherein said surfactant is a non-ionic surfactant. 
     
     
         10 . The method of  claim 9 , wherein said non-ionic surfactant is selected from the group consisting of a POE polymer, POP polymer, POE-POP block copolymer, PEG polymer, Brij® surfactant, Igepal® surfactant, Tween® surfactant and Triton® surfactant. 
     
     
         11 . The method of  claim 1 , wherein said surfactant in said Solution S2 varies in concentration from about 0.001% to about 10%. 
     
     
         12 . The method of  claim 11 , wherein said surfactant in said Solution S2 varies in concentration from about 0.005% to about 1%. 
     
     
         13 . The method of  claim 4 , wherein said surfactant in said Solution S1 varies in concentration from about 0.001% to about 10%. 
     
     
         14 . The method of  claim 13 , wherein said surfactant in said Solution S1 varies in concentration from about 0.005% to about 1%. 
     
     
         15 . The method of  claim 1 , wherein said Solution S2 further comprises a PEG polymer. 
     
     
         16 . The method of  claim 15 , wherein said PEG polymer varies in concentration in said Solution S2 from about 0.1% to about 10%. 
     
     
         17 . The method of  claim 16 , wherein said PEG polymer varies in concentration in said Solution S2 from about 1% to about 8%. 
     
     
         18 . The method of  claim 4 , wherein said Reagent R1 further comprises a PEG polymer. 
     
     
         19 . The method of  claim 18 , wherein said PEG polymer varies in concentration in said Solution S1 from about 0.1% to about 10%. 
     
     
         20 . The method of  claim 19 , wherein said PEG polymer varies in concentration in said Solution S1 from about 1% to about 8%. 
     
     
         21 . The method of  claim 1 , wherein said Solution S2 optionally further comprises one or more components selected from the group consisting of PEG, dextran sulfate, α-cyclodextrin sulfate, EDTA, an azide salt, and a divalent cation. 
     
     
         22 . The method of  claim 4 , wherein said Reagent R1 optionally further comprises one or more components selected from the group consisting of PEG, dextran sulfate, α-cyclodextrin sulfate, EDTA, an azide salt, and a divalent cation. 
     
     
         23 . The method of  claim 4 , wherein said Reagent R2 optionally further comprises one or more components selected from the group consisting of PEG, dextran sulfate, α-cyclodextrin sulfate, EDTA, an azide salt, and a divalent cation. 
     
     
         24 . The method of  claim 1 , wherein said Solution S2 comprises a composition selected from the group consisting of: (i) surfactant Pluronic® F-68 (0.5%), PEG 4000 (2.5%), and PBS buffer, pH 7.4; (ii) surfactant Brij® 700 (0.005%), PEG 8000 (3%), and PBS buffer, pH 7.4; and, (iii) Pluronic® F-127 (0.01%), PEG 8000 (5%), and PBS buffer, pH 7.4. 
     
     
         25 . The method of  claim 4 , wherein said Solution S1 comprises a composition selected from the group consisting of: (i) surfactant Pluronic® F-68 (0.5%), PEG 4000 (2.5%), and PBS buffer, pH 7.4; (ii) surfactant Brij® 700 (0.005%), PEG 8000 (3%), and PBS buffer, pH 7.4; and, (iii) Pluronic® F-127 (0.01%), PEG 8000 (5%), and PBS buffer, pH 7.4. 
     
     
         26 . The method of  claim 1 , wherein said period of time t2 is about 3 minutes and said set temperature T2 is about 37° C. 
     
     
         27 . The method of  claim 4 , wherein said period of time t1 is about 5 minutes and said set temperature T1 is about 37° C. 
     
     
         28 . The method of  claim 1 , wherein said immunoassay is immunoturbidimetry or immunonephelometry. 
     
     
         29 . The method of  claim 1 , wherein said immunoassay is an ELISA assay. 
     
     
         30 . The method of  claim 1 , wherein said at least one standard solution has said known concentration expressed in mg/dL units. 
     
     
         31 . The method of  claim 1 , wherein said concentration of said lipoprotein-apolipoprotein in said biological fluid is calculated in mg/dL units. 
     
     
         32 . The method of  claim 1 , wherein said concentration of said lipoprotein-apolipoprotein in said biological fluid is calculated in nmoles/L units. 
     
     
         33 . The method of  claim 1 , wherein said concentration of said lipoprotein particles in said biological fluid is calculated in nmoles/L units. 
     
     
         34 . A composition comprising a non-ionic surfactant selected from the group consisting of a POE polymer, a POP polymer, a POE-POP block copolymer, a PEG polymer, a Brij® surfactant, a Igepal® surfactant, a Tween® surfactant and a Triton® surfactant, wherein the concentration of said non-ionic surfactant varies from about 0.001% to about 10%; in a buffer with a pH value ranging from about 6.5 to about 8.5%; said composition optionally further comprising a PEG polymer varying in concentration from 0.1% to about 10%; and optionally further comprising one or more components selected from the group consisting of PEG, dextran sulfate, α-cyclodextrin sulfate, EDTA, an azide salt and a divalent cation. 
     
     
         35 . The composition of  claim 34 , further comprising a biological fluid from a subject. 
     
     
         36 . The composition of  claim 34 , further comprising an antibody against an apolipoprotein. 
     
     
         37 . A kit for measuring the concentration of lipoprotein particles in a biological fluid of a subject, wherein an apolipoprotein is structurally associated with said lipoprotein in the form of lipoprotein-apolipoprotein, said kit comprising:
 at least one composition comprising a known amount of said apolipoprotein,   a composition comprising a non-ionic surfactant selected from the group consisting of a POE polymer, a POP polymer, a POE-POP block copolymer, a PEG polymer, a Brij® surfactant, a Igepal® surfactant, a Tween® surfactant and a Triton® surfactant, wherein the concentration of said non-ionic surfactant varies from about 0.001% to about 10%; in a buffer with a pH value ranging from about 6.5 to about 8.5; said composition optionally further comprising a PEG polymer varying in concentration from about 0.1% to about 10%; and optionally further comprising one or more components selected from the group consisting of PEG, dextran sulfate, α-cyclodextrin sulfate, EDTA, an azide salt and a divalent cation;   a composition comprising an antibody against said apolipoprotein and optionally further comprising one or more components selected from the group consisting of PEG, dextran sulfate, α-cyclodextrin sulfate, EDTA, an azide salt and a divalent cation;   
       said kit further comprising an applicator and an instructional material for the use of said kit.